DESCRIPTION: Cocaine is a significant cofactor for HIV-1 infection, transmission and morbidity. Cocaine accelerates HIV-1 gene expression by altering specific cell-signaling and epigenetic pathways and promotes HIV-associated neurocognitive disorders (HAND). Our published data demonstrates that cocaine-induced signaling pathways lead to the activation of kinases, such as MSK1 which subsequently catalyzes the phosphorylation of p65 subunit of NF- ?B at position S276 (P-p65S276) and histone H3 at Serine 10 (P-H3S10). P-H3S10, besides facilitating the establishment of transcriptionally active chromatin structures, promotes the recruitment of P-TEFb at HIV LTR. Consequently, cocaine accelerates both the initiation and the elongation phases of HIV transcription by activating NF-kB and P-TEFb, respectively, a prerequisite to generate complete HIV genomic transcript and new viral progeny. However, our understanding of the signaling pathways that cocaine utilizes in order to promote these changes and induce enzymes, including MSK1/2, RSK 1/2, Aurora-B, ELK1 and IKK-? is highly obscure. These enzymes directly and indirectly induce different epigenetic modifications, which never been investigated in the context of HIV infection. Thus, it is imperative to have clear knowledge about the cocaine induced signaling pathways that affect HIV replication. In this grant we have proposed a systematic analysis. First, we will define and characterize the cocaine induced signaling pathways that lead to the activation of above mentioned different kinases, their target proteins and their gene regulation. Later, we will define the role of cocaine induced epigenetic modifications and the precise role of involved enzymes and their effect on HIV gene expression and replication. Subsequently, we will define the signaling pathways which cocaine induces to activate NF-kB and P-TEFb. Finally, we will utilize high throughput approaches to characterize involved epigenetic modifications and the genes which are influenced by cocaine exposure; in order to find unique biomarkers of cocaine use. All the results will be reproduced in monocytic/macrophage and microglial cell lines. Crucial findings will be confirmed using primary Monocyte derived macrophages (MDMs) from cocaine using HIV patients. Finally, to validate the direct effect of cocaine, we will perform ex vivo experiments where we will expose PBMCs from HIV infected patients to cocaine. Broader Impact: These studies will identify many novel actions of cocaine, involved pathways and enzymes which can be exploited for the development of improved diagnostic tests and may open up new avenues for better pharmaceutical interventions in drug addict HIV patients.